The anthrax toxin lethal factor (LF) enzyme directly enables the Bacillus anthracis bacterium to evade host immunological mechanisms, leading to circulatory shock and death. Few, if any, therapeutic options are available to counteract LF-mediated cytotoxicity at any stage of anthrax infection. The long-term goal is to better understand the mechanisms by which anthrax toxins act, and, based upon that knowledge, to develop novel and relevant biochemical tools that can be utilized to define mechanisms of LF-related cell death and tissue damage. The objective in this particular application is to evaluate new, promising molecular scaffolds for efficacy against LF-mediated toxicity. The central hypothesis is that small molecules structurally related to a new glutamic acid-based scaffold will effectively inhibit LF protease activity in vitro and in cell-based assays. The rationale for the proposed research is that an improved understanding of key structural features that contribute to LF inhibition will, in turn, lead to an improved understanding of toxin involvement in anthrax pathogenesis, and will potentially provide a promising strategy for treating postexposure anthrax. Thus, the proposed research is relevant to that part of NIH's mission that pertains to developing fundamental knowledge that can be applied to advance significantly the Nation's capacity to protect and improve health. Guided by strong preliminary data, the central hypothesis will be tested by pursuing four specific aims: 1) Design, synthesize and evaluate new glutamic acid-based libraries targeting the anthrax toxin lethal factor active site; 2) Identify new scaffolds for LF probe design using high-throughput screening; 3) Identify probe compounds with capacity to protect macrophages against LF-mediated cytotoxicity; and 4) Identify key binding modes to the LF enzyme active site by means of X-ray crystallography. Under the first aim, libraries around candidate scaffolds will be generated in silico; compounds will be prioritized using molecular modeling; and selected compounds will be synthesized and evaluated for activity against LF in vitro. In the second aim, a large-scale high-throughput screen with novel triage and hit-to-probe techniques will be conducted to identify additional potential scaffolds for probe design; refinement from hit scaffolds will follow an iterative, systematic cycle of design, synthesis, purification, and screening. In the third aim, probes will be tested to assess their relative protective efficacy in inhibiting cell death of a murine macrophage cell line in response to anthrax toxin; and in the fourth aim, prioritized compounds will be co-crystallized with LF in order to experimentally identify key LF ligand-receptor interactions. The approach is innovative, because it targets specific LF structural features that have not been fully investigated, and incorporates recently developed, highly accurate computational and experimental techniques not yet applied to the LF system. The proposed research is significant, because it is expected to provide further molecular insights into the pathways leading to LF-related cell death, and in so doing, to advance and expand understanding of the complex mechanisms involved in anthrax toxemia. PUBLIC HEALTH RELEVANCE: The proposed work addresses important and under-investigated molecular structures and mechanisms that contribute to anthrax pathogenesis. The proposed research has relevance to public health, because although weaponized anthrax continues to pose a threat to society, there is currently no effective therapeutic on the market that can counteract LF-mediated cell death, and the findings from this work are ultimately expected to guide the design of effective therapeutics that can aid persons who have been, or suspect they may have been, exposed to anthrax spores in an emergency situation.